The Role of LPS in the Opioid Issue
We need to become aware of the role of LPS, the very common bacterial toxin, in creating the opioid
problem for children with autism. Scientific articles prove that LPS produces leaky gut , damaged vili, brain body barrier permiability and opioid sensitivity: all the conditions that make the children with autism more vulnerable to the opioid effect. Moreover LPS induces an increase in beta-endorphin, a natural opioid made by the body.
The evidence for the role of LPS is so thick that even the first article that demonstrated that children with autism are sensitive to gliadin, soy and dairy mentioned LPS.
That famous experiment found a correlation between the children's sensitivity for gliadin, soy and dairy and their reactions to LPS. The authors suggest that the root cause of the food protein sensitivity may be an underlying aberrant innate immune response against LPS.
The dairy free version of SCD, the version of SCD most often used by parents of ASD children, helps the opioid issue in autism by eliminating soy, gluten and cow's milk, the most likely foods to produce an opioid effect. However,
it is important to note that soy, gluten and cow's milk are not the only sources of opioids for children with autism. Other foods such a spinach contain small amounts of opioids. Moreover, children with autism
have been found to have a significant excess of beta-endorphin, a natural opioid made by the body.
It is interesting to note that many children with autism begin to tolerate the goat yogurt after several months of SCD. This might be due to the improvement of the opioid situation after the removal of bacterial toxins.
Conclusion: Children with autism need to eliminate bacterial toxins in order to completely be free from abnormal opioid levels.
Article showing that spinach contains an opioid peptide.
Click here to view this article on PubMed
1: FEBS Lett. 2001 Dec 7;509(2):213-7.
Rubiscolin, a delta selective opioid peptide derived from plant Rubisco.
Yang S, Yunden J, Sonoda S, Doyama N, Lipkowski AW, Kawamura Y, Yoshikawa M.
Division of Food Bioscience and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
We found that the sequences YPLDL and YPLDLF in the large subunit of spinach D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) met the structure YP-aliphatic amino acid which might have opioid activity. We then synthesized these peptides to test their opioid activity. The IC(50) of these peptides in mouse vas deferens assay were 51.0 microM and 24.4 microM, respectively, and those in delta receptor binding assay using [(3)H]deltorphin II as radioligand were 2.09 microM and 0.93 microM, respectively. Both peptides were selective for delta receptor. We named them rubiscolin-5 and -6, respectively. Rubiscolin-5 and -6 have antinociceptive activity in mice after i.c.v. or oral administration. The enzymatic conditions to release rubiscolin were investigated using both spinach Rubisco and synthetic fragment peptides. This is the first example of bioactive peptides derived from plant Rubisco.
PMID: 11741591 [PubMed - indexed for MEDLINE]
Beta-endorphin is significantly elevated in children with autism
Click here to view this article on PubMed
1: Pediatr Neurol. 1987 Mar-Apr;3(2):83-6.
Reduction of elevated CSF beta-endorphin by fenfluramine in infantile autism.
Ross DL, Klykylo WM, Hitzemann R.
Department of Neurology, University of Cincinnati, Ohio.
Fenfluramine therapy has been reported to improve behavior in infantile autism and has been associated with a decrease in abnormally increased blood serotonin content. The primary central effect has not been proved to be serotonergic. Beta-endorphin is involved in the anorexic effect of fenfluramine and may play a role in autism. Nine children with infantile autism were treated with fenfluramine in double-blind, placebo-crossover design. Transient anorexia was the only adverse effect. Autistic behavior was reported to improve in three patients, but objective psychometric tests were unchanged. Beta-endorphin-like immunoreactivity was determined in lumbar cerebrospinal fluid of patients during and before or after treatment with fenfluramine and then was compared to normal controls. Beta-endorphin was elevated significantly in the baseline autistic group (p less than .005) and was reduced toward control values during fenfluramine treatment. The results are consistent with a role for beta-endorphin in infantile autism and in the mechanism of fenfluramine treatment.
PMID: 2977280 [PubMed - indexed for MEDLINE]
The drug, Fenfluramine, was withdrawn from the U.S. market in 1997 after reports of heart valve disease, and pulmonary hypertension, including a condition known as cardiac fibrosis. After the US withdrawal of fenfluramine, it was also withdrawn from other markets around the world. (From Wikipedia)
LPS causes intestinal injuries that result in a leaky gut.
The next article shows that LPS causes the tight junctions to widen and become disrupted.
Please note that this alteration plays an important role in creating a leaky gut. The other changes induced by LPS include broken microvilli, a sign of celiac disease.
View this article in PubMed
1: Zhongguo Dang Dai Er Ke Za Zhi. 2006 Oct;8(5):425-8. Links
[Protective effects of recombinant intestinal trefoil factor against intestinal injuries induced by endotoxin in young rats]
[Article in Chinese]
* Li J,
* Xu LF,
* Sun M,
* Li Q,
* Gao H,
* Jiang WG.
Department of Pediatrics, Second Affiliated Hospital of China Medical University, Shenyang 110004, China. firstname.lastname@example.org
OBJECTIVE: This study aimed to investigate the protective effects of recombinant intestinal trefoil factor (rITF) against intestinal injuries and the possible mechanism by examining the changes of diamine oxidase (DAO) and TNF-alpha and the intestinal ultrastructural changes in lipopolysaccharide (LPS) induced intestinal injuries. METHODS: Ninety-six ten-day-old Wistar rats were randomly injected with either normal saline (1 mL/kg, Control group), LPS (1 mL/kg) or LPS (1 mL/kg) + rITF (0.1 mL) intraperioneally. At 2, 6, 24 and 72 hrs after administration plasma DAO activity was determined using absorption spectrometry; and the intestinal protein and mRNA expression of TNF-alpha were measured using immunohistochemistry and RT-PCR methods. The intestinal ultrastructural changes were observed by electron microscopy. RESULTS: The plasma DAO activity in the LPS group began to increase at 2 hrs, peaked at 6 hrs and remained at significantly higher levels until 72 hrs after administration compared with the Control group (P < 0.01). The plasma DAO activity in the LPS + rITF group decreased noticeably compared with the LPS group at all time points (P < 0.01 or 0.05). A significant difference in the plasma DAO activity was only observed at 6 hrs after administration between the LPS + rITF and the Control group. The expression of TNF-alpha protein in the LPS group significantly increased at each time point, peaking at 6 hrs after LPS administration, with the IODT of TNF-alpha of 37,247.64 +/- 3,387.59 vs 6,191.02 +/- 482.32 (P < 0.01) compared with the Control group. rITF treatment decreased the expression of TNF-alpha protein although it remained significantly higher than in the Control group (P < 0.01). The TNF-alpha mRNA was weakly expressed in the Control group but strikingly increased after LPS injection (P < 0.01). Compared with the LPS group, the TNF-alpha mRNA expression in the LPS + rITF group decreased at all time points (P < 0.01 or 0.05). Vacuole changes of mitochodrium, cell nucleus condense, break and depletion of part of microvilli, and widen and disrupted tight junction were observed in the LPS group. The ultrastructural changes of intestinal tissues were improved in the LPS + rITF group. CONCLUSIONS: rITF can decrease the plasma DAO activity and inhibit the expression of TNF-alpha, resulting in a protective effect against intestinal injuries induced by LPS in young rats.
PMID: 17052407 [PubMed - indexed for MEDLINE]
Another research article that demonstrates that endotoxin (another name for LPS)
causes intestinal mucosal permeability (another name for leaky gut) This article also uses the term lipopolysaccharide for LPS.
Click here to view this article on PubMed
The effect of endotoxin on intestinal mucosal permeability to bacteria in vitro.
* Go LL,
* Healey PJ,
* Watkins SC,
* Simmons RL,
* Rowe MI.
Department of Pediatric Surgery, Children's Hospital of Pittsburgh, Pa.
OBJECTIVE: To examine the role of the intestinal mucosa in bacterial translocation, in vitro bacterial passage across ileal mucosal segments mounted in Ussing chambers were studied in control and endotoxin (lipopolysaccharide)-treated rats. DESIGN: Experimental study. MATERIALS AND METHODS: Three groups of rats were studied. The experimental group received an intraperitoneal injection of lipopolysaccharide, while controls received an equivalent volume of saline solution; a third group received no treatment. Twenty-four hours later, all groups underwent laparotomy and organ culture to assess bacterial translocation. At the same time, a segment of mucosa from the terminal ileum of each animal was mounted in a Ussing chamber, and the transmucosal passage of labeled Escherichia coli from the luminal to serosal surface was assessed by results of serial cultures. RESULTS: In vivo bacterial translocation occurred in 100% of the lipopolysaccharide-treated animals, significantly higher than the incidence seen in controls (25%; P < .05). In vitro passage of labeled E coli across ileal mucosa in the Ussing chamber occurred in 78% of lipopolysaccharide-treated animals, while in controls transmucosal passage was seen in only 14% (P < .05). Histologic examination of mucosa from both groups using light and transmission electron microscopy demonstrated no structural differences between groups. CONCLUSIONS: Increased permeability to bacteria at the mucosal level contributes to the bacterial translocation seen in endotoxemia.
PMID: 7802577 [PubMed - indexed for MEDLINE]
View this article in PubMed
Long-term changes in blood-brain barrier permeability and white matter following prolonged systemic inflammation in early development in the rat.
* Stolp HB,
* Dziegielewska KM,
* Ek CJ,
* Potter AM,
* Saunders NR.
Department of Pharmacology, University of Melbourne, Parkville, VIC 3010, Australia.
Epidemiological evidence in human fetuses links inflammation during development with white matter damage. Breakdown of the blood-brain barrier has been proposed as a possible mechanism. This was investigated in the present study by inducing a prolonged inflammatory response in newborn rats, with intraperitoneal injections of lipopolysaccharide (LPS; 0.2 mg/kg) given at postnatal (P) day 0, P2, P4, P6 and P8. An acute phase response was present over the whole period of injections. Changes in blood-brain barrier permeability were determined for small (sucrose and inulin) and large (protein) molecules. During and immediately after the inflammatory response, plasma proteins were detected in the brain only within white matter tracts, indicating an increased permeability of the blood-brain barrier to protein during this period. The alteration in permeability to protein was transient. In contrast, the permeability of the blood-brain barrier to 14C-sucrose and 14C-inulin was significantly higher in adult animals that had received serial LPS injections during development. Adult animals receiving a single 1 mg/kg LPS injection at P0 showed no alteration in blood-brain barrier permeability to either small or larger molecules. A significant decrease in the volume of CNPase immunoreactive presumptive white matter tracts occurred in the external capsule and corpus callosum at P9. These results demonstrate that a prolonged systemic inflammatory response in the early postnatal period in rats causes size selective increases in blood-brain barrier permeability at different stages of brain development and results in changes in white matter volume.
PMID: 16324115 [PubMed - indexed for MEDLINE]
Articles showing that LPS stimulates the release of beta-END (beta-endorphin).
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1: Neuroendocrinology. 1994 Sep;60(3):243-51.
Role of hypothalamic histaminergic neurons in mediation of ACTH and beta-endorphin responses to LPS endotoxin in vivo.
Knigge U, Kjaer A, Jørgensen H, Garbarg M, Ross C, Rouleau A, Warberg J.
Department of Medical Physiology, Panum Institute, University of Copenhagen, Denmark.
The involvement of hypothalamic histaminergic neurons in the mediation of the ACTH and beta-endorphin (beta-END) response to lipopolysaccharide (LPS) endotoxin was investigated in conscious male rats. LPS stimulated the release of ACTH and beta-END dose-dependently and increased the hypothalamic concentration of the histamine (HA) metabolite tele-methylhistamine significantly and that of HA slightly, indicating an increased turnover of neuronal HA. Pretreatment with the HA synthesis inhibitor alpha-fluoromethyl-histidine administered intracerebroventricularly (i.c.v.) or intraperitoneally (i.p.) inhibited the ACTH and beta-END response to LPS about 60%, whereas i.p. administration of the H3 receptor agonist R(alpha)methylHA, which inhibits HA synthesis and release, decreased the response about 50%. Pretreatment with the H1 receptor antagonist mepyramine (67 micrograms x 2 i.c.v.) inhibited the hormone response to LPS about 50%, while pretreatment with equimolar doses of the H2 receptor antagonists cimetidine (67 micrograms x 2 i.c.v.) or ranitidine (83 micrograms x 2 i.c.v.) had no effect on the LPS-induced release of ACTH and beta-END. When the H1 receptor antagonists mepyramine and cetirizine were administered i.p. in doses (10 mg/kg) which penetrate the blood-brain barrier the hormone response to LPS was inhibited 50% and 30%, respectively. Administered i.p. the H2 receptor antagonists had no effect on the hormone response to LPS. We conclude that hypothalamic histaminergic neurons in rats are involved in the mediation of the ACTH and beta-END response to LPS stimulation via activation of central postsynaptic H1 receptors.
PMID: 7969782 [PubMed - indexed for MEDLINE]
Click here to view on PubMed.
1: Crit Care Med. 1992 Mar;20(3):402-8.
Endorphin mediation of mesenteric blood flow after endotoxemia in sheep.
Navaratnam N, Herndon DN, Woodson LC, Linares HA, Morris S, Traber DL.
Department of Anesthesiology, University of Texas Medical Branch, Galveston.
BACKGROUND AND METHODS: The administration of endotoxin in small dosages to sheep results in an acute decrease followed by an increase in cardiac output. It has previously been determined that the initial decrease in output was the result of a reduction in blood flow to the mesenteric areas. These changes were associated with increased blood concentrations of beta endorphin. The present study was accomplished to determine if the systemic cardiovascular response to endotoxin could be affected by the administration of an opiate receptor-blocking agent. Female range sheep (n = 12) were prepared for chronic study by implantation of cardiopulmonary catheters and a flow probe on the cephalic mesenteric artery. Endotoxin (Escherichia coli, 1 microgram/kg) was administered to these animals. Half of the sheep were treated with naloxone (2 mg/kg + 2 mg/kg.hr), and the remainder with an equivalent volume of sodium chloride (0.9%). RESULTS: In untreated sheep, cardiac indices decreased by 15% to 20% (5.1 +/- 0.1 to 4.2 +/- 0.4 L/min.m2) between 0 and 1 hr and 2 and 5 hrs after endotoxin (4.5 +/- 0.2 L/min.m2), and then increased to a value 40% (7.2 +/- 0.6 L/min.m2) above baseline by 12 hrs. Flow in the cephalic mesenteric artery decreased in a pattern similar to the reduction in cardiac index (962 +/- 152 [time, T = 0] to 379 +/- 111 [T = 0.8] and 384 +/- 88 mL/min [T = 4.0], p less than .05) but did not increase to the same extent (1008 +/- 153 mL/min [T = 4.0], p greater than .05). There was a concomitant increase in the plasma beta-endorphin concentrations as the blood flow decreased (5 +/- 4 [T = 0] to 40 +/- 15 pg/mL [T = 0.8; untreated group], p less than .05; and 10 +/- 4 to 50 +/- 7 pg/mL [T = 0.8; naloxone-treated group], p less than .05). In the naloxone-treated group, the same pattern of cardiac output change was noted with endotoxin; however, reduction of mesenteric artery flow was only 30% (1118 +/- 129 to 908 +/- 122 mL/min, p less than .05) of the value seen in the untreated animals (962 +/- 152 to 379 +/- 111 mL/min, p less than .05). These changes in mesenteric blood flow were statistically significant from one another. As the cardiac output increased in the sheep treated with the opiate antagonist, mesenteric blood flows increased 20% above the baseline value (1391 +/- 199 mL/min, p less than .05). CONCLUSIONS: The decrease in cardiac output noted with endotoxin can be accounted for by the decrease in the blood flow in the cephalic mesenteric artery. This phenomenon can be attributed, at least in part, to the release of endorphins. There is both a vasodilation and constriction during endotoxemia in the ovine model.
PMID: 1541102 [PubMed - indexed for MEDLINE]
LPS and opioid sensitivity.
For many people in the autism community, the opioid effect is at the heart of the explanation of ASD. Research indicates that LPS induces an opioid effect: analgesia(a lack of pain. ) This resembles the effects from morphine
Definition of Analgesic and its relation to morphine and other opiates.
View an article about analgesia and its relation to morphine
Research showing that LPS induces an opioid effect:
View this article in PubMed
1: Brain Res. 1994 Jun 13;648(1):80-6.
Behavioral effects of lipopolysaccharide in rats: involvement of
* Yirmiya R,
* Rosen H,
* Donchin O,
* Ovadia H.
Department of Psychology, Hebrew University of Jerusalem, Mount
Activation of the immune system in response to either infection or
lipopolysaccharide (LPS) produces neurophysiological, neuroendocrine
and behavioral changes.
Some of the physiological consequences of
LPS are mediated by endogenous opioid peptides. The following
studies were designed to characterize the effects of LPS in several
behavioral paradigms, and to determine the role of opioids in
mediating these effects. The effects of LPS on locomotor and
self-care activity were assessed in the open field test. Rats were
injected with either saline or a dose of LPS (25, 50, 100, or 1000
micrograms/kg). 4 h later, the animals were placed in an open field
and the numbers of line crossings, rearings and grooming episodes
were counted. LPS significantly suppressed the three open field
behaviors in a dose-related manner. The effect of LPS on sensitivity
to pain was determined using the hot-plate and tail-flick tests.
Administration of LPS (200 micrograms/kg) increased pain sensitivity
in the hot plate test 30 min after drug administration, but produced
a significant analgesic response 4 h after drug administration in
both tests. Further characterization of LPS-induced analgesia
demonstrated that it began about 2 h after and disappeared 30 h
after the administration of LPS. Administration of naltrexone
completely blocked the analgesic effects of LPS 4 h after its
administration, but had no effect on LPS-induced suppression of
activity in the open field. The effect of LPS on body temperature
was biphasic, producing hypothermia at 2 h and hyperthermia at 8-30
h after its administration. Naltrexone had no effect on the body
temperature changes induced by LPS.(ABSTRACT TRUNCATED AT 250 WORDS)
PMID: 7922530 [PubMed - indexed for MEDLINE]
LPS increases tendency for addiction and might might precipitate alcohol drinking behavior.
We know that many kids with ASD tend to have food addiction.
View this article in PubMed
Endotoxin exposure in utero increases ethanol consumption in adult male offspring.
* Liu X,
* Lee JG,
* Yee SK,
* Bresee CJ,
* Poland RE,
* Pechnick RN.
Department of Psychiatry, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. email@example.com
Epidemiological studies have suggested that adverse experiences in utero predispose individuals to neurobehavioral disorders including drug abuse in adulthood. The present study was designed to examine the hypothesis that maternal endotoxin exposure during pregnancy increases ethanol consumption in adult offspring. Pregnant Sprague-Dawley rats were subjected to lippopolysaccharide (LPS, 1.0 mg/kg, s.c.) treatment on alternate days throughout pregnancy. Adult male offspring were tested for ethanol consumption by using a free-access and two bottle choice paradigm. The animals exposed to LPS showed increased ethanol intake and preference as well as decreased rearing activity in the open field test. These data suggest that maternal infection during pregnancy might precipitate alcohol drinking behavior in adult offspring and this effect might be due, at least in part, to elevated levels of anxiety.
PMID: 15106858 [PubMed - indexed for MEDLINE]
Click here to view this article on PubMed
1: Curr Gastroenterol Rep. 1999 Oct;1(5):410-6.
Intestinal permeability, leaky gut, and intestinal disorders.
Harbor-UCLA Research and Education Institute, 1124 West Carson Street, Torrance, CA 90502, USA.
A major task of the intestine is to form a defensive barrier to prevent absorption of damaging substances from the external environment. This protective function of the intestinal mucosa is called permeability. Clinicians can use inert, nonmetabolized sugars such as mannitol, rhamnose, or lactulose to measure the permeability barrier or the degree of leakiness of the intestinal mucosa. Ample evidence indicates that permeability is increased in most patients with Crohn's disease and in 10% to 20% of their clinically healthy relatives. The abnormal leakiness of the mucosa in Crohn's patients and their relatives can be greatly amplified by aspirin preadministration. Permeability measurements in Crohn's patients reflect the activity, extent, and distribution of the disease and may allow us to predict the likelihood of recurrence after surgery or medically induced remission. Permeability is also increased in celiac disease and by trauma, burns, and nonsteroidal anti-inflammatory drugs. The major determinant of the rate of intestinal permeability is the opening or closure of the tight junctions between enterocytes in the paracellular space. As we broaden our understanding of the mechanisms and agents that control the degree of leakiness of the tight junctions, we will be increasingly able to use permeability measurements to study the etiology and pathogenesis of various disorders and to design or monitor therapies for their management.
PMID: 10980980 [PubMed - indexed for MEDLINE]
LPS is linked to the problems of gluten,soy and dairy in ASD children.
A famous experiment with ASD children found a correlation between the sensitivity for gliadin,soy and dairy and the children's reactions to LPS. The authors suggest that the root cause of the food protein sensitivity may be an underlying sensitivity to endotoxin or lipopolysaccharides (LPS) which comes from the surfaces of gram negative bacteria in the gut.
This is a very important since the elimination of gliadin,soy and dairy is the backbone of the GFCF diet. (gliadin is A glycoprotein (a carbohydrate plus a protein) within gluten. )
Below is the article by Jyonouchi that links LPS to dairy,gliadin and soy sensitivity.
Elaine Gottschall's Comments on the Jyonouchi Paper
The abstract of Jyonouchi's paper says that children with autism spectrum disorder (ASD) frequently reveal various gastrointestinal (GI) symptoms that may resolve with an elimination diet along with apparent improvement of some of the behavorial symptoms. Evidence suggests that ASD may be accompanied by aberrant (inflammatory) innate immune responses. THIS MAY PREDISPOSE ASD CHILDREN TO SENSITIVIES TO COMMON DIETARY PROTEINS LEADING TO GI INFLAMMATION AND AGGRAVATION OF SOME BEHAVIORAL SYMPTOMS."
The authors demonstrate conclusively that there is an abnormal immune response to cow's milk protein and wheat protein (gliadin) and soy in ASD. What is interesting is that the diet model is NOT THE SAME AS THE OPIOID MODEL BUT IS BASED ON A VARIABLE IMMUNE RESPONSE IN WHICH NOT EVERY CHILD WILL SHOW SENSITIVITY TO EVERY FOOD. But the punch line is: The authors suggest that the root cause of the food protein sensitivity may be an underlying sensitivity to endotoxin or lipopolysaccharides (LPS) which comes from the surfaces of gram negative bacteria in the gut.
Summary: This response to the bacterial endotoxin (LPS) PREDISPOSES THESE CHILDREN TO SENSITIZATION TO DIETARY PROTEINS. This is consistent with a model of abnormal gut flora development that promotes immune response to gut bacteria. This means that ASD kids may develop a kind of autoimmunie response to their own gut flora.
(Some of these comments were written by Mark Blaxhill.)
The Jyonouchi Paper
Innate immunity associated with inflammatory responses and cytokine production against common dietary proteins in patients with autism spectrum disorder.
Jyonouchi H, Sun S, Itokazu N.
Department of Pediatrics, University of Minnesota, Minneapolis, Minn, USA.
OBJECTIVES: Children with autism spectrum disorder (ASD) frequently reveal various gastrointestinal (GI) symptoms that may resolve with an elimination diet along with apparent improvement of some of the behavioral symptoms. Evidence suggests that ASD may be accompanied by aberrant (inflammatory) innate immune responses. This may predispose ASD children to sensitization to common dietary proteins (DP), leading to GI inflammation and aggravation of some behavioral symptoms.
METHODS: We measured IFN-gamma, IL-5, and TNF-alpha production against representative DPs [gliadin, cow's milk protein (CMP), and soy] by peripheral blood mononuclear cells (PBMCs) from ASD and control children [those with DP intolerance (DPI), ASD siblings, and healthy unrelated children]. We evaluated the results in association with proinflammatory and counter-regulatory cytokine production with endotoxin (LPS), a microbial product of intestinal flora and a surrogate stimulant for innate immune responses. RESULTS: ASD PBMCs produced elevated IFN-gamma and TNF-alpha, but not IL-5 with common DPs at high frequency as observed in DPI PBMCs.
ASD PBMCs revealed increased proinflammatory cytokine responses with LPS at high frequency with positive correlation between proinflammatory cytokine production with LPS and IFN-gamma and TNF-alpha production against DPs. Such correlation was less evident in DPI PBMCs. CONCLUSION: Immune reactivity to DPs may be associated with apparent DPI and GI inflammation in ASD children that may be partly associated with aberrant innate immune response against endotoxin, a product of the gut bacteria. Copyright 2002 S. Karger AG, Basel
PMID: 12378124 [PubMed - indexed for MEDLINE]